Journal
Journal of Applied Horticulture, 8(2): 117-120, July-December, 2006
Appl
Effects of sugars and aminooxyacetic acid on the
longevity of pollinated Dendrobium (Heang Beauty) flowers
S. Chandran1, C.L. Toh1, R. Zuliana1, Y.K. Yip1, H. Nair2 and A.N. Boyce1
Institute of Biological Science, University of Malaya, 50603 Kuala Lumpur; 2 Asian Institute of Medicine, Science and
Technology, 2 Persiaran Cempaka, Amanjaya, 0800 Sungai Petani, Kedah Darul Aman, Malaysia.
E-mail: chandran@um.edu.my
1
Abstract
The vase life of detached pollinated Dendrobium (Heang Beauty) orchids are affected by loss of energy source and the production
of pollination-induced ethylene. The physiological changes that follow these two events are discoloration, thinning of petals and
hyponasty. In order to circumvent this problem, individual detached pollinated Dendrobium (Heang Beauty) flowers were treated
with solutions containing different concentrations of sucrose or glucose, Aminooxyacetic acid (AOA) and a combination of sugars
and AOA. Discolouration, petal thickness and hyponasty were observed and data was recorded daily. Weight loss of flowers and pH
of all solutions were also measured daily. Results showed that the best treatment solution in extending the longevity of the flowers
were solutions containing 4% sucrose + 0.5mM AOA. Flowers held in this treatment also showed a delay in discoloration, thinning
of petals and hyponasty. The inclusion of AOA into solutions resulted in low pH and contributes to better water uptake and delayed
turgor loss in flowers.
Key words: Dendrobium, pollination, ethylene, AOA, flowers, discoloration, sucrose, glucose
Introduction
The cut flower industry is a US$ 2 billion industry and is an
important revenue for some of the major cut flower producers in
the world. Orchids are among the highly demanded cut flowers
and are produced by countries like Thailand, the Netherlands,
Hawaii, Singapore and Italy. However, like any other cut flower,
orchids face vase life-related problems such as excessive water
loss, decline in respirable substrates and sensitivity to exogenous
or endogenous ethylene that hastens senescence and wilting of
the flowers (Hew, 1994).
Vase-life is a yardstick for the longevity of cut flowers and is an
important target for improving flower characteristics, whether
by chemical treatment or plant breeding (Yamada et al., 2002).
The influence and effect of sugars and ethylene are two major
areas which have been vastly studied to tackle the problem of
flower quality and vase life of cut flowers.
Several studies have focused on the effect and mechanism of
ethylene inhibitors. Compounds such as aminooxyacetic acid
(AOA), aminoethoxyvinylglycine (AVG) and silver thiosulphate
(STS) effectively delay senescence of climacteric flowers by
inhibiting the synthesis or action of 1-aminocyclopropane-1
carboxylate synthase (ACCS) (Yang, 1984). Petal senescence is
generally accelerated by treatment with exogenous ethylene and
delayed by anionic STS, which prevents ethylene action (Beyer,
1976). For example, gladiolus flowers were reported to last longer
(Hwang et al., 1995) when they were treated with silver STS.
Furthermore, the use of sugars in holding solution of cut flowers
has been extensively studied and has yielded a great amount of
success in a variety of cultivars. Inclusion of sugars in the vase
solution of carnation flowers reduced their sensitivity to ethylene
(Mayak and Dilley, 1976). Sucrose, that extended vase-life of
rose (Kuiper et al., 1995) and sweet pea (Ichimura and Hiraya,
1999), also extended vase-life of gladiolus (Marousky, 1971;
Bravdo et al., 1974). From these observations, it is clear that
sugars like sucrose have a negative effect on the process of cell
death leading to petal senescence.
This study aims to further establish the effectiveness of AOA
and sugars in prolonging the vase life of pollinated Dendrobium
(Heang Beauty).
Materials and method
Plant material: Dendrobium (Heang Beauty) orchids were taken
from the glasshouse in the University of Malaya. Flowers were
cut at the peduncle, and were hand-pollinated by removing the
pollinia from the anther to the stigma on the same flower using
forceps. The cut flowers were held in glass vials containing 22ml
of distilled water (control) or solutions containing chemicals. It
was important for the peduncle of the flowers to be immersed
in the solutions to enable efficient uptake of water and nutrient.
The weight of the flowers were between 1.24-1.27g. All flowers
were held at room temperature (26 ± 4oC).
Ethylene measurements: Ethylene production of detached
unpollinated and pollinated Dendrobium flowers was measured
using a Hewlett-Packard Gas Chromatography every 12 hours.
Flowers were weighed as individual flowers were held in 30 mL
glass centrifuge tube throughout the experiment. Tubes were
sealed for 2 hours before readings were taken. After each reading,
the seals were removed until the next time point.
Preparation of chemicals: AOA (Sigma) was tested at
concentration of 0.25 and 0.5mM. Glucose (Sigma) and sucrose
(Sigma) were used at 4%. All solutions were prepared at the
beginning of the experiments and were not renewed.
pH readings: The pH of the solutions was determined daily with
a pH meter (HANNA Instruments).
Water uptake and fresh weight: The difference between
consecutive weighing of the vial plus solution (without the
flower) was used to calculate the water uptake. Evaporative water
loss from the surface of the solution was negligible. The weight
of each flower was determined daily by subtracting the weight of
the vial and solution from weight of vial, solution and flower.
Measurement of thickness: Thickness of each flower was
measured daily by using a micrometer (Mitutoyo Micrometer).
Flower wilting: Vase life of flowers was considered terminated
when the flowers reached full closure. The experiment was run
against a flower treated with distilled water as control.
Colour measurement: The colour of petals was measured daily
using a Minolta CR-200 Chromameter using the L.a.b Munsell
Colour System. L* indicated value, a* and b* measures hue
and chroma. Colour change was calculated using L*, a* and b*
values and the formula: L* x a* / b*
Results
In this experiment, ethylene production of detached pollinated
Dendrobium flowers were measured against unpollinated
Dendrobium flowers to confirm the climacteric response in
the flowers. As shown in Fig. 1, pollinated flowers produced
ethylene at 60 hours (1.25 μg g-1h-1), 84 hours (1.4 μg g-1h-1) and
108 hours (1.3 μg g-1h-1) after pollination . Thereafter the ethylene
production decreased and remained at 0 μg g-1h-1. Unpollinated
flowers (control) did not produce any ethylene throughout the
experiment.
Pollinated Dendrobium held in distilled water (control), had
the shortest vase life (3 days) compared to pollinated flowers
in the other treatments. Flowers treated with 0.5mM AOA, 4%
glucose with 0.5 mM AOA and 4% sucrose with 0.5 mM AOA
proved to be the better treatments in extending longevity (Table
1). The combination of 4% sucrose and 0.5mM AOA was the
best treatment because of its ability to extend vase life up to 13
days.
Table 1. Vase life of pollinated Dendrobium (Heang Beauty) held in
different treatments
Treatment
Full closure (day)
Distilled water
6 ±0.3
Glucose 4%
8 ±0.5
Sucrose 4%
7 ±1.0
0.25mM AOA
7 ±0.5
0.5mM AOA
11 ±0.3
Sucrose 4% + 0.5mM AOA
13 ±0.5
Glucose 4% + 0.5mM AOA
11 ±0.0
Generally, all the treatments showed weight increment in the
first 3 days and began to decrease thereafter. The increment
may be due to the initial water uptake from the solutions to fulfil
the nutrient requirement of the cut flowers. However, at day 3,
the water uptake was no longer effective to increase the fresh
weight of the flowers and the weight began to drop. The fresh
weight became lower when the flowers started to close (result not
shown). Control showed a drop in weight of flowers at day 2 and
the rapid loss of weight continued throughout the experiment. On
Amount of ethylene ( g g-1 h-1)
Effects of sugars and aminooxyacetic acid on the longevity of pollinated Dendrobium
1.6
1.4
1.2
1
Unpollinated
Pollinated
0.8
0.6
0.4
0.2
0
0
24
48
72
96
120
144
Time(hour)
Fig. 1. Ethylene production of unpollinated and pollinated detached
Dendrobium Heang Beauty.
1.6
1.4
1.2
weight (g)
118
1
0.8
Distilled water
0.5 mM AOA
4% glucose+0.5 mM AOA
4% sucrose+0.5 mM AOA
0.6
0.4
0.2
0
0
1
2
3
4
5
6
7
8
Time (d)
9
10
11
12
13
14
Fig. 2. Weight of detached pollinated Dendrobium Heang Beauty held
in different treatment solutions.
the other hand, flowers treated with 0.5mM AOA, sucrose 4% +
0.5mM AOA and glucose 4% + 0.5mM AOA managed to delay
the onset of weight loss to day 4 and day 5 with a more stable
weight loss throughout the experiment (Fig. 2).
Table 2 shows the total water uptake of Dendrobium flowers held
in different treatments throughout the treatment. Flowers held in
4% sucrose +0.5mM AOA showed the highest amount of water
uptake (4.46 mL), followed by flowers held in 0.5mM AOA (3.94
mL) and flowers held in 4% glucose+ 0.5mM AOA (3.83 mL).
Total water uptake by the flowers held in the rest of the treatment
solutions was not significantly different from the control.
Table 2. Total water uptake by pollinated Dendrobium (Heang Beauty)
flowers held in different treatments
Treatments
Distilled water
Glucose 4%
Sucrose 4%
0.25mMAOA
0.5mM AOA
Sucrose 4%+0.5mMAOA
Glucose 4%+0.5mMAOA
Total water uptake (mL)
2.00
2.17
2.19
2.65
3.94
4.46
3.83
Fig. 3 shows the change in thickness of flower petals of pollinated
Dendrobium throughout the experiment. In this observation, the
positive results coincided with the longevity of flowers. This is
evident as flowers treated with 0.5mM AOA, 4% glucose with 0.5
mM AOA and 4% sucrose with 0.5 mM AOA managed to show
a delay in decrement of petal thickness which only occurred at
day 6 (4% sucrose + 0.5mM AOA) and day 4 (0.5mM AOA, 4%
glucose) compared to control which showed decrease in petal
thickness on day 1.
Effects of sugars and aminooxyacetic acid on the longevity of pollinated Dendrobium
0.06
Thickness (mm)
into three types as follows:
Distilled water
0.5 mM AOA
4% glucose+0.5 mM AOA
4% sucrose+0.5 mM AOA
0.05
0.04
(i)Type I, wilting apparently mediated by ethylene;
(ii)Type II, wilting apparently not mediated by ethylene;
(iii)Type III, abscission apparently mediated by ethylene.
0.03
0.02
0.01
0
0
2
4
6
8
10
12
14
Time (d)
Fig. 3. Thickness of petals of pollinated Dendrobium (Heang Beauty)
flowers held in different treatment solutions.
80
L* x a*/b*
60
50
40
30
20
10
0
0
2
4
6
8
Time (days)
10
12
In the cut flower species showing Types I and III of petal
senescence, i.e. ethylene-sensitivity, vase-life could be improved
by treatment with ethylene inhibitors or by genetic transformation
with ethylene related genes (Reid and Wu, 1992; Chang et al.,
1993). The Dendrobium falls under the category of flowers
where the senescence and abscission are mediated by ethylene.
Furthermore this particular flower also shows pollination induced
ethylene production which eventually acts as a signal for the
flower to undergo senescence-like physiological changes.
Inhibitors of ethylene production and action have shown to slow
down floral abscission (Sexton et al., 1985). Aminooxyacetic
acid (AOA) is an inhibitor of pyridoxal phosphate-requiring
enzymes including 1-aminocyclopropane-1-carboxylic acid
(ACC) synthase, a key enzyme of ethylene synthesis (Abeles et
al., 1992). The results from this experiment further establishes
the effectiveness of AOA as an ethylene inhibitor as the treatment
with 0.5 mM AOA resulted in a significant extension of vase life
compared to that of the control.
Distilled water
0.5 mM AOA
4% glucose+0.5 mM AOA
4% sucrose+0.5 mM AOA
70
119
14
Fig. 4. Colour change of petals of pollinated Dendrobium (Heang
Beauty) flowers held in different treatment solutions.
The decrease in L.a.b value is an indication of discoloration
of petal, from white to eventually transparent. Flowers held in
0.5mM AOA, 4% glucose+0.5mM AOA and 4% sucrose+0.5mM
AOA showed a lower rate of discolouration of petals compared
to the control (Fig. 4).
Table 3 shows the change in pH values of the different treatments.
It was found that the pH of solutions containing AOA (0.025 mM
AOA, 0.5 mM AOA, sucrose 4% +0.5mM AOA and glucose +
with 0.5mM AOA) were maintained around 3 throughout the
experiment. Treatments with sugars however, showed higher pH
values (5.0-6.0) throughout the experiment. The pH values of the
solutions directly influence the water relations of the flowers.
Table 3. pH value of treatment solutions of at day 0 and day of
termination of the experiment
Treatment
pH of solution at
pH of
initial wilting
solution
at day of
termination
Distilled Water
4.8+0.01
6.2+0.02
4% Glucose
4.2+0.02
5.3+0.01
4% Sucrose
4.4+0.02
5.8+0.02
0.25 mM AOA
3.6+0.03
3.8+0.03
0.5 mM AOA
3.1+0.01
3.5+0.01
4% Glucose + 0.5 mM AOA
3.3+0.04
3.4+0.04
4% Sucrose + 0.5 mM AOA
3.1+0.02
3.3+0.02
Discussion
It is a well known fact that the effect of ethylene on wilting can
be inhibited by ethylene inhibitors. Woltering and van Doorn
(1988), classified petal senescence of 93 species from 22 families
The post harvest life of flowers is strongly dependent on the
carbohydrate status and the acceptable amounts of metabolic
sugars are factors that affect the rate of senescence. Keeping
flowers in vase solutions containing sucrose has been shown
to extend their vase-life (Ho and Nichols, 1977). Supplying cut
flowers with exogenous sugar maintain the pool of dry matter
and respirable substrates, especially in petals, thus promoting
respiration and extending longevity (Coorts, 1973; Rogers,
1973).
Aarts (1957) suggested that exogenous sucrose in some way
maintains the structure and semi-permeability of the plasma
membrane. It was demonstrated that sucrose antagonized
the effect of abscisic acid in promoting the senescence of
roses (Borochov et al., 1976). Furthermore, treatments of cut
flowers with sucrose are found to be beneficial in delaying
senescence processes (Yakimova et al., 1996). Exogenous
supply of sugars delays wilting in many flowers and this effect
is due to maintenance in starch and sugar levels in cut flowers
(Rattanawisalanon et al., 2003).
Previous studies have found that sucrose interact with other
internal plant hormones in regulating the process of senescence.
It was shown that sucrose enhances the effect of cytokinins in
delaying senescence of flowers and reduces the effect of ethylene
in promoting it (Mayak and Dilley, 1976). Sugar is found to be
involved in enhancing the function of certain organelle such
as mitochondria. Kaltaler and Steponkus (1976) found that in
mitochondria isolated from cut flowers pretreated with sucrose,
respiratory control values were maintained over longer periods.
Hence, they concluded that the main effect of applied sugar in
extending longevity is to maintain mitochondrial structure and
functions. All of these findings show a close relationship with the
results and effect of sugars observed in this experiment.
However, the main disadvantage of sugars in the vase solution
is the promotion of bacterial growth when not accompanied by
120
Effects of sugars and aminooxyacetic acid on the longevity of pollinated Dendrobium
an adequate antimicrobial agent; and it may therefore clog the
xylem vessels and inhibit the uptake of both water and dissolved
sugars (van Doorn, 1997). As is shown from the results, the
effectiveness of sugar as a continuous energy supply for cut
flowers was retarded due to the absence of an antimicrobial agent
causing stem blockage. Hence, the turgidity and overall quality
of the vase life was not significantly maintained throughout the
experiment.
Rattanawasilanon et al. (2003) found that inclusion of AOA in
the vase water, together with a sugar, had a positive effect on
the time to flower senescence. In pollinated flowers, the effect
of AOA on wilting may be due to a reduction of endogenous
ethylene synthesis; however, such an effect is present only when
AOA is combined with sugars (Rattanawasilanon et al., 2003).
Treatments with AOA or AOA + sucrose effectively retarded
the longevity of cut spray-carnation flowers (Yakimova et al.,
1997). Our result is in agreement with both these findings as the
combination of AOA and sucrose showed the longest vase life
of pollinated Dendrobium.
AOA therefore, can act as an antibacterial agent, which inhibits
bacterial growth (Rattanawasilanon et al., 2003). The ability of
AOA as an antimicrobial agent is attributed to the maintenance
of low pH which results in a non-conducive environment
for bacterial growth. Low pH seems to play the role of an
antibacterial agent as bacterial growth is virtually halted at a
pH of three or lower (Ketsa and Narkbua, 2001). Water uptake
was also enhanced by acidic (pH 3 to 4) and warm water (43oC)
(Dole and Schnelle, 2002). Therefore, solutions with low pH
throughout the experiment (0.5mM AOA. sucrose 4% + 0.5mM
AOA) considerably delayed both abscission and petal wilting as
water relations were improved and maintained.
In this experiment post pollination symptoms observed were
discolouration of petals, change in petal thickness, weight loss
and changes in pH values. Sucrose 4% with 0.5mM AOA was
proved to be the best chemical treatment in delaying the petal
senescence of Dendrobium (Heang Beauty). The treatment
was able to maintain the highest water uptake by the flowers
throughout the experiment. The lowest rate of thickness decrement
of this treatment indicated their ability to slow down the pectin
hydrolysis of petals throughout the experiment. It remains to be
the centre of future studies to investigate the consequences of
different ethylene inhibitors for their contribution in prolonging
the vase life of cut flowers.
References
Aarts, J.F.T. 1957. On the keepability of cut flowers. Meded.
Landbouwhogesch. Wageningen 57: 1-62.
Abeles, F.B. 1973. Ethylene in plant biology. Academic Press, New
York. pp 136-142.
Beyer, E.M.J. 1977. Ethylene: its incorporation and oxidation to carbon
dioxide by cut carnations. Plant Physiol., 60: 203-206.
Borochov, A., S. Mayak and A.H. Halevy, 1976. Combined effects of
acid and sucrose on growth and senescence of rose flowers. Plant
Physiol., 36: 221-224
Bravdo, B., S. Mayak and Y. Gavijel, 1974. Sucrose and water uptake
from concentrated sucrose solutions by gladiolus shoots and the
effect of these treatments on floret life. Can. J. Bot., 52: 12711281.
Chang, C., S.F. Kwok, A.B. Bleecker and Meyerowitz, 1993.
Arabidopsis ethylene-response gene ETR1: similarity of product
to two-component regulators. Science, 262: 539-544.
Coorts, G.D. 1973. Internal metabolic changes in cut flowers.
HortScience, 8: 195.
Dole, M.J. and M.A. Schnelle, 2002. The care and handling of cut
flowers. Division of Agricultural Sciences and Natural Resources,
Oklahoma State University.
Hew, C.S. 1994. “Orchid cut- flower production in ASEAN countries”
In: Orchid Biology: Reviews and Perspectives, Vol VI, ed. J. Arditti
(John Wiley and Son Inc, New York) pp. 363-401.
Ho, L. and R. Nichols, 1977. Translocation of 14C-sucrose in relation
to changes in carbohydrate content in rose corollas cut at different
stages of development. Ann. Bot., 41: 227-242.
Hwang, M.J. and K.S. Kim, 1995. Postharvest physiology and
prolonging vase life of cut gladiolus. J. Kor. Soc. Hort. Sci., 36(3):
410-419.
Ichimura, K. and T. Hiraya, 1999. Effect of silver thiosulfate complex
(STS) in combination with sucrose on the vase life of cut sweet
pea flowers. J. Japan. Soc. Hort. Sci., 68: 23-27.
Kaltaler, R.E.L. and P.L. Steponkus, 1976. Factors affecting respiration
in cut roses. J. Amer. Soc. Hort. Sci., 101: 352-354.
Ketsa, S. and N. Narkbua, 2001. Effect of Aminooxyacetic acid and
sucrose on vase life of cut roses. Acta Hortic., 543: 227-234.
Kuiper, D., S.A. Ribot, H.S. Van Reenen and N. Marissen, 1995. The
effect of sucrose on the flower bud opening of Madelon cut roses.
Sci. Hortic., 60: 325-336.
Marousky, F.J. 1971. Influence of 8-hydroxyquinoline citrate and
sucrose on carbohydrate content of leaves and florets of cut
gladiolus spikes. Acta Hortic., 23:127-131.
Mayak S. and D.R. Dilley, 1976. Effect of sucrose on response of cut
carnation to kinetin, ethylene and abscissic aid. Am. Soc. Hort.
Sci., 101: 583-585.
Rattanawisalanon, C., S. Ketsa and W.G. van Doorn, 2003. Effect of
aminooxyacetic acid and sugars on the vase life of Dendrobium
flowers. Postharvest Biology and Technology, 29: 93-100
Reid, M.S. and M.J. Wu, 1992. Ethylene and flower senescence. Plant
Growth Regul., 11: 37-43.
Rogers, M. 1973. An historical and critical review of post harvest
physiology research on cut flowers. HortScience, 8: 189-194.
Sexton, R., L.N. Lewis, A.J. Trewavas and P. Kelly, 1985. “Ethylene and
abscission” In: Ethylene and Plant Development. J.A. Roberts and
G.A.Tucker (Eds). Butterworths, Boston, MA, USA, pp. 173-196
Van Doorn, W.G. 1997. Effect of pollination on floral attraction and
longevity. Journal of Experimental Botany, 48: 1615-1622
Woltering, E.J. and W.G. Van Doorn, 1988. Role of ethylene in
senescence of petals-morphological and taxonomical relationships.
J. Exp. Bot., 208: 1605-1616.
Yakimova, E., B.B. Atanassova and V. Kapchina-Toteva, 1997.
Longevity and some metabolic events in post-harvest Spraycarnation flowers. Bulg. J. Plant Physiol., 23: 57-65.
Yamada, T., Y. Takatsu, T. Manabe, M. Kasumi and W. Marubashi,
2003. Suppressive effect of trehalose on apoptotic cell death leading
to petal senescence in ethylene-insensitive flowes of gladiolus. Plant
Science, 164: 213-221.
Yang S.F. and N.E. Hoffman, 1984. Ethylene biosynthesis and its
regulation in higher plants. Annu. Rev. Pl. Physiol., 35: 155-189.